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Published online before print February 1, 2006, 10.1110/ps.051917406
Protein Science (2006), 15:635-639. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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FOR THE RECORD

Monitoring protein aggregation during thermal unfolding in circular dichroism experiments

Sangeeta Benjwal1,3, Shikha Verma2,3, Klaus-Heinrich Röhm2 and Olga Gursky1

1 Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118, USA
2 Institute of Physiological Chemistry, Philipps University, Marburg 35043, Germany

(RECEIVED October 18, 2005; FINAL REVISION November 16, 2005; ACCEPTED November 21, 2005)

Thermal unfolding monitored by spectroscopy or calorimetry is widely used to determine protein stability. Equilibrium thermodynamic analysis of such unfolding is often hampered by its irreversibility, which usually results from aggregation of thermally denatured protein. In addition, heat-induced protein misfolding and aggregation often lead to formation of amyloid-like structures. We propose a convenient method to monitor in real time protein aggregation during thermal folding/ unfolding transition by recording turbidity or 90° light scattering data in circular dichroism (CD) spectroscopic experiments. Since the measurements of turbidity and 90° light scattering can be done simultaneously with far- or near-UV CD data collection, they require no additional time or sample and can be directly correlated with the protein conformational changes monitored by CD. The results can provide useful insights into the origins of irreversible conformational changes and test the linkage between protein unfolding or misfolding and aggregation in various macromolecular systems, including globular proteins and protein–lipid complexes described in this study, as well as a wide range of amyloid-forming proteins and peptides.

Keywords: circular dichroism spectroscopy; irreversible protein unfolding; turbidity; light scattering; asparaginase-2; high-density lipoprotein; amyloid; protein structure/folding; conformational changes; stability and mutagenesis; enzymes; thermodynamics; hydrodynamics; aggregation

Abbreviations: CD, circular dichroism • DSC, differential scanning calorimetry • EcA2, asparaginase isoenzyme 2 from E. coli • apoC-1, apolipoprotein C-1 • DMPC, dimyristoyl phosphatidylcholine

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051917406.

Reprint requests to: Olga Gursky, Department of Physiology and Biophysics, Boston University School of Medicine, W329, 715 Albany Street, Boston, MA 02118, USA; e-mail: Gursky{at}bu.edu; fax: (617) 638-4041.


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